Long QT syndrome (LQT) is a cardiac condition characterized by prolonged ventricular repolarization, increasing the risk of life-threatening arrhythmias. It can be inherited due to mutations in genes like HERG, which encodes the pore-forming subunit of the rapid delayed rectifier K⁺ current (Iₖr), or acquired as a side effect of medications that block HERG channels. This study investigates the structural basis for drug-induced block of HERG channels, focusing on MK-499, a methanesulfonanilide antiarrhythmic drug. Using alanine-scanning mutagenesis, the researchers identified key residues in the S6 transmembrane domain (G648, Y652, F656) and pore helix (T623, V625) of HERG that interact with MK-499. These residues face the channel cavity and are crucial for high-affinity drug binding. Other drugs, such as terfenadine and cisapride, also interact with Y652 and F656 but not V625. These aromatic residues are unique to eag/erg K⁺ channels, unlike other voltage-gated K⁺ channels (Kv1–Kv4), which have Ile and Val in equivalent positions. This structural difference explains why many drugs block HERG but not other Kv channels. The study also shows that mutations in these residues significantly affect drug sensitivity. Homology modeling confirmed that MK-499 binds to the channel cavity, interacting with aromatic residues through π-stacking and electrostatic interactions. The findings suggest that the high-affinity binding site for methanesulfonanilides is the inactivated state of HERG. This structural insight could aid in designing drugs that avoid HERG channel binding, reducing the risk of drug-induced LQT. The study highlights the importance of understanding the molecular mechanisms of drug-channel interactions to improve drug safety and efficacy.Long QT syndrome (LQT) is a cardiac condition characterized by prolonged ventricular repolarization, increasing the risk of life-threatening arrhythmias. It can be inherited due to mutations in genes like HERG, which encodes the pore-forming subunit of the rapid delayed rectifier K⁺ current (Iₖr), or acquired as a side effect of medications that block HERG channels. This study investigates the structural basis for drug-induced block of HERG channels, focusing on MK-499, a methanesulfonanilide antiarrhythmic drug. Using alanine-scanning mutagenesis, the researchers identified key residues in the S6 transmembrane domain (G648, Y652, F656) and pore helix (T623, V625) of HERG that interact with MK-499. These residues face the channel cavity and are crucial for high-affinity drug binding. Other drugs, such as terfenadine and cisapride, also interact with Y652 and F656 but not V625. These aromatic residues are unique to eag/erg K⁺ channels, unlike other voltage-gated K⁺ channels (Kv1–Kv4), which have Ile and Val in equivalent positions. This structural difference explains why many drugs block HERG but not other Kv channels. The study also shows that mutations in these residues significantly affect drug sensitivity. Homology modeling confirmed that MK-499 binds to the channel cavity, interacting with aromatic residues through π-stacking and electrostatic interactions. The findings suggest that the high-affinity binding site for methanesulfonanilides is the inactivated state of HERG. This structural insight could aid in designing drugs that avoid HERG channel binding, reducing the risk of drug-induced LQT. The study highlights the importance of understanding the molecular mechanisms of drug-channel interactions to improve drug safety and efficacy.